The present invention is in the field of bone physiology and morphology, and specifically, describes the identification and use of selected bisphosphonates and calcitonin derivatives to increase bone mass which (i) inhibit the apoptosis of osteocytes and/or osteoblasts (ii) without substantially affecting the activity of osteoclasts.
Bones consist of living cells embedded within a matrix of proteins and minerals. Bones provide support and protection to the vital organs of the animal, and give strength and form to its structure. Diseases of the bone, therefore, may have significant deleterious effects on humans as well as other vertebrates.
Osteoporosis is a decrease in bone mass in combination with microarchitectural deterioration which leads to bone fragility and fractures. Treatments for osteoporosis have historically focused on the prevention of further bone loss. In contrast, a bone anabolic agent is one that substantially increases bone mass. An increase in bone mass does not necessarily lead to a decrease in bone fragility. To date, while there have been several drugs approved by the U.S. Food and Drug Administration for the treatment of osteoporosis, it is believed that no drug has yet been approved in the United States to be used as a bone anabolic agent, for either humans or other animals.
Bone is a dynamic tissue which undergoes continual resorption and formation through a remodeling process, which is accomplished by two types of cells: osteoclasts, which erode cavities, and osteoblasts that synthesize new bone matrix. Remodeling takes place mainly on the internal surfaces of bone and it is carried out not by individual cells, but rather by temporary anatomical structures, termed basic multi-cellular units (BMUs), comprising teams of osteoclasts in the front and osteoblasts in the rear. In an established BMU, bone resorption and formation happens at the same time.
After osteoclasts stop resorbing bone, they die by apoptosis and are quickly removed by phagocytes. During the longer lifespan of the osteoblasts (about three months, as compared to three weeks for osteoclasts), some osteoblasts convert to lining cells that cover quiescent bone surfaces and some are entombed within the mineralized matrix as osteocytes (Parfitt, In: Bone, Telford and CRC Press, PP351-429, 1990). However, the majority (65%) of osteoblasts that originally assembled at the remodeling site die by apoptosis (Jilka et al., JBMR 13:793-802, 1998).
Osteocytes are the most abundant bone cell type and are buried deep in the mineralized bone matrix within lacunae connected with canaliculi through which the long and slender cytoplasmic processes of osteocytes are connected with neighboring osteocytes and with the cells on the bone surface and of the bone marrow.
Because of their sheer number, regular spacing throughout the mineralized matrix and their anatomical connections with other bone cells, osteocytes are believed to be the sensors of the local need for bone augmentation or reduction during functional adaptation of the skeleton, the detection of microdamage, and the transmission of signals that lead to bone repair by remodeling. Specifically, it is thought that mechanical strains on bone cause deformations that result in flow of fluid within the osteocytic lacunae and canaliculi. The changes in fluid flow are sensed by the osteocytes, which, in turn, transmit signals to osteoblasts (new bone-forming cells) and osteoclasts (old bone-removing cells). Osteoblasts and osteoclasts react by remodeling the mineral tissue so that it is permanently adapted to daily mechanical deformations. When this system fails, the tissue becomes fragile, and bone structure proves inadequate and brittle.
Bone fragility is a pathologic condition that may be caused by various factors, including a poor quality of mineralized tissue or more usually by weak structure, unable to respond competently to the customary mechanical requirements of the skeleton. Poor osteocytic activity is related to this state of fragility (Duncan R L et al., Calcif. Tissue Int. 1995, 57:344; Mullender M G et al., Bone 1997, 20:527; Turner C H et al., Bone 1998, 22:463). The bone structures that jointly constitute the human skeleton and that of vertebrate animals are permanently distorted by the application of external forces, in which muscular force usually intervenes (Ferretti J L et al., Calcif. Tissue Int. 1995, 57:399; Frost H M, Bone 1997, 20:385). Consistent with the critical role of osteocytes in bone maintenance, it has recently been demonstrated that glucocorticoid excess, traditionally a cause of severe boneloss and osteonecrosis leading to the collapse of joints, dramatically increases osteocyte and osteoblast apoptosis (Weinstein et al., J. Clin. Invest., 102:274); whereas, intermittent administration of parathyroid hormone, a method of anabolic bone therapy, has the opposite effect on osteocyte and osteoblast apoptosis (Jilka et al., J. Clin. Invest., 104:439-446, 1999).
Most metabolic disorders of the adult skeleton result from an imbalance between the resorption of old bone by osteoclasts and its subsequent replacement by osteoblasts. Changes in cell numbers, opposed to individual cell activity (Manolagas and Jilka, NEJM 332:305-311, 1995), appears to be the cause of most metabolic bone diseases, including the three most common forms of osteoporosis: osteoporosis due to sex steroid deficiency in females and males (Jilka et al., Science 257:88-91, 1992; Jilka et al., JCI 101:1942-1950, 1998; Bellido et al., JCI 95:2886-2895, 1995; Weinstein et al., Endocrinology 138:4013-4021, 1997); osteoporosis due to old age (Jilka et al., JCI 97:1732-1740, 1996); and osteoporosis due to glucocorticoid-excess (Weinstein et al., JCI 102:274-282, 1998; Weinstein et al., Bone, 23:S461, 1998; Bellido et al., Bone, 23:S324, 1998). Structural bone alterations caused by decreased osteocyte life span predisposes the bone to irreversible deformations and fractures. This condition is designated xe2x80x9cskeletal fragility.xe2x80x9d
Agents that reduce bone turnover by inhibiting remodeling (commonly but inaccurately referred to as xe2x80x9cantiresorptivexe2x80x9d) increase bone mass by a maximum of 6-10%, and more typically, 2-3%, as measured by Dual Energy X-Ray Absorptiometry (DEXA). Most of this increase is in the first 1-2 years and is due to contraction of the remodeling space. Modest further increases may result from more complete secondary mineralization. Improvement of focal balance due to reduction of resorption depth has been demonstrated in animal experiments, but not yet in human subjects. Regardless of the mechanism, an increase of less than 10% will in almost all cases fail to restore bone mass to its peak value and fail to reestablish trabecular connectivity so that fracture risk will remain increased.
Over the past three decades, bisphosphonates (BP""s), stable analogs of pyrophosphate and calcitonin have been developed as potent inhibitors of bone resorption and effective agents for the management of osteoporosis and other bone diseases (Fleisch, H. 1997. Bisphosphonates in bone disease. From the laboratory to the patient. The Partenon Publishing Group Inc., One Blue Hill Plaza, New York 10965, USA.; Papapoulos, S. 1996. Bisphosphonates. Pharmacology and use in the treatment of osteoporosis. In Osteoporosis. R. Marcus, D. Feldman, and J. Kelsey, editors. Academic Press, San Diego, Calif. 1209-1234 Rodan, G. A. and H. A. Fleisch. 1996. J. Clin. Invest. 97:2692-2696; Azria, et al., 1996. Calcitonin. In Principles of Bone Biology. J. P. Bilezikian, et al., eds, Academic Press, San Diego, Calif. 1083-1097). Decreased osteoclast progenitor development, decreased osteoclast recruitment, and promotion of apoptosis of mature osteoclasts leading to decreased bone remodeling are thought to be the main mechanisms of the antiresorptive actions of BPs (Hughes, et al., 1995. J. Bone Miner. Res. 10:1478-1487; Hughes, et al., 1989. J. Clin. Invest. 83:1930-1935; Parfitt, et al., 1996. J. Bone Miner. Res 11:150-159). Likewise, disruption of osteoclast function is the main mechanism for the antiresorptive actions of calcitonin (Azria, et al., 1996. Calcitonin. In Principles of Bone Biology. J. P. Bilezikian, et al., eds, Academic Press, San Diego, Calif. 1083-1097). At least some of the effects of BPs on osteoclast development and function might be mediated indirectly through actions on cells of the osteoblastic lineage. Thus, pretreatment of osteoblastic cells with BPs inhibits the formation of osteoclast-like cells from their marrow or spleen precursors (Nishikawa, et al., 1996. Bone 18:9-14), as well as osteoclast resorbing activity in concultures with mature osteoclasts (Sahni, et al., 1993. J. Clin. Invest. 91:2004-2011; Vitte, et al., 1996. Endocrinology 137:2324-2333). These inhibitory effects can be reproduced by addition of conditioned media from BP-treated osteoblastic cells to the cultures, suggesting that BPs promote the release of factors that inhibit osteoclast formation and activity (Nishikawa, et al., 1996. Bone 18:9-14; Sahni, et al., 1993. J. Clin. Invest. 91:2004-2011; Vitte, et al., 1996. Endocrinology 137:2324-2333).
Long-term treatment of human and nonhuman primates with BPs increases wall thickness, an index of focally increased osteoblast numbers or activity, resulting in more complete refilling of resorption cavities (Chavassieux, et al., 1997. J. Clin. Invest. 100:1475-1480; Storm, et al., 1993. J. Bone Miner. Res. 8:199-208.). This evidence has raised the possibility that BPs do more than simply reduce remodeling space and that they may possess anabolic activity (Giuliani, et al., 1998. Bone 22:455-461).
Antiresorptive agents such as BPs and calcitonin, as well as estrogen, decrease fracture incidence disproportionally to their effect on bone mass (Cummings, et al., 1996. J. Bone Miner. Res. 11 (suppl):S102(Abstr.)). This suggests an additional effect on bone strength unrelated to effects on bone mineral density (BMD). However, an explanation for this phenomenon has remained elusive. Osteocytes, differentiated osteoblasts regularly spaced throughout the mineralized matrix, are believed to detect bone microdamage and to transmit signals leading to its repair (Marotti, et al., 1990. Ital. J. Min. Electrol. Metab. 4:93-106; Nijweide et al., 1996, The osteocyte, Principles of Bone Biology, Bilezikian, et al. editors, Academic Press, San Diego, Calif. 115-126.). Disruption of the osteocyte network might compromise this mechanism, leading to microdamage accumulation and increased bone fragility. Such a defect in bone quality might account for the higher incidence of fractures in glucocorticoid-treated patients compared with postmenopausal women, even though BMD in the former is relatively higher (Peel, et al., 1995. Ann. Rheum. Dis. 54:801-806; Dennison, E. 1999. Epidemiology of glucocorticoid-induced osteoporosis. Osteoporosis Int. 9:S16(Abstr.)). There have been observations that glucocorticoid excess increases the prevalence of osteocyte and osteoblast apoptosis (Weinstein, et al., 1998. Bone 23(suppl):S461(Abstr.); Weinstein et al., 1998. J. Clin. Invest. 102:274-282). and that BPs are effective in the management of this condition (Reid, et al., 1990. J. Bone Miner. Res. 5:619-623; Gonnelli, et al., 1997. Calcif. Tissue Int. 61:382-385; Falcini, et al. 1996. Calcif. Tissue Int. 58:166-169).
The currently approved treatments for osteoporosis focus on inhibiting osteoclastic bone resorption. Inhibition of osteoclastic bone resorption causes less removal of xe2x80x9coldxe2x80x9d mineral tissue and avoids excessive losses of calcified structures, such as those which occur in certain skeletal diseases, particularly osteoporosis. Although treatment with bisphosphonates allows for preservation of a greater quantity of skeletal mass, such withholding may slow down or disturb the adjustment of the skeleton by xe2x80x9cfreezingxe2x80x9d tissue renewal. Moreover, by means of the anti-osteoclast treatment, incompetent mineralized structures may be preserved and thus cause xe2x80x9cfatiguexe2x80x9d to the neighboring structures. xe2x80x9cFatiguexe2x80x9d happens with all overburdened material structures, paradoxically increasing the risk of fracture.
Approved therapeutic agents for osteoporosis, such as bisphosphonates, estrogen and calcitonin, are believed to exert their beneficial effects by inhibiting osteoclastic bone resorption. There are currently ten classes of drugs that are used in the treatment of osteoporosis: anabolic steroids, bisphosphonates, calcitonins, estrogens/progestogens, Selective Estrogen Receptor Modulators (SERMs) such as raloxifene and phytoestrogen, parathyroid hormone (xe2x80x9cPTHxe2x80x9d), fluoride, Vitamin D metabolites, and calcium preparations. No compound within these classes has been approved as a bone anabolic agent.
Anabolic Steroids (Androgens)
Anabolic steroids (androgens) have bee n known to build muscle mass in the host. However, there has been no reported evidence that they function as bone anabolic agents as defined herein (Snyder et al., JCEM 84:1966-1972, 1999). Androgens are typically used as a replacement therapy for male hypogonadal disorders and they are used in adolescent males with a history of delayed puberty or growth. Androgens can produce significant side effects when taken over a period of time, including water retention, jaundice, decreased high density lipoprotein and increased low density lipoprotein, hepatic toxicity (most usually associated with the 17xcex1-alkylated androgens), hepatic carcinoma, increased risk of cardiovascular disease, and when taken in large dosages, irrationality, psychotic episodes, violent behavior, and death. U.S. Pat. No. 5,565,444 discloses the use of an androgen for the treatment of bone loss or for increasing bone mass.
Calcitonin
Endogenous calcitonin is a polypeptide hormone involved in the regulation of calcium and bone metabolism. Forms used therapeutically include calcitonin (pork), extracted from pig thyroid, a synthetic human calcitonin; elcatonin, a synthetic analogue of eel calcitonin; and salcatonin, a synthetic salmon calcitonin. They all have the property of lowering plasma-calcium concentration by diminishing the rate of bone resorption. Calcitonins are typically administered subcutaneously or by intramuscular injection.
Bisphosphonates
Bisphosphonates, as stated above, have been widely used to treat osteoporosis. The bisphosphonate disodium etidronate has similar effects on bone mass and fractures in established osteoporosis to those of calcitonin, but cannot be given for a prolonged period because of the risk of osteomalacia. Bisphosphonate alendronate treatment at a dose of 10 mg/day results in a 5% increase in spinal bone mineral density (BMD) over the first year (Dempster, Exploiting and Bypassing the Bone Remodeling Cycle to Optimize the Treatment of Osteoporosis, Journal of Bone and Mineral Research, Volume 12, Number 8, 1997, pages 1152-1154). BMD continues to increase, albeit at a slower rate, at this site during the second and third years of treatment. The magnitude and duration of the increase in BMD has led to speculation that alendronate is doing more than simply reducing remodeling space and that it may possess anabolic activity. The bisphosphonate etidronate reduced resorption depth in human iliac trabecular bone by almost 30% after one year of treatment, but no such data are yet available for alendronate. Etidronate did not change the thickness of trabecular packets, but recent studies in osteoporotic women suggest that this is increased after two years of alendronate treatment at 10 and 20 mg/day. This result was not confirmed after three years of treatment.
In another article, Dempster (Dempster D. W., New concepts in bone remodeling, In: Dynamics of Bone and Cartilage Metabolism, Chapter 18, pp. 261-273, Acad. Press, 1999) confirms that the potential for an agent that can increase bone mass and hence reverse the skeletal defect in patients with osteoporosis is great, particularly if in doing so it also repairs microarchitectural damage. He notes that estrogens and calcitonin primarily stabilize bone mass and prevent further loss of bone, although a transient small increment in mass is often reported, particularly in patients with elevated levels of bone remodeling. Dempster et al. conclude that this is not a true anabolic effect but is related to the temporal effects on turnover in which resorption declines initially followed by a reduction in formation that may take several months.
It was disclosed in the priorty document to this application, for the first time, that that bisphosphonates have anti-apoptotic effects on osteoblasts and osteocytes. This fundamental discovery has been published by the inventors after the priority date in Plotkin et al. Bone, 23:S157, 1998; J. Clin. Invest; 104:1363-1374, 1999. Significantly, the anti-apoptotic effect of bisphosphonates in vitro is achieved with doses 100-1000 lower than the doses at which these same agents inhibit osteoclast activity; and additionally can be demonstrated with bisphosphonates that do not block osteoclast activity at all (compound IG9204).
U.S. Pat. No. 4,870,063 discloses a bisphosphonic acid derivative to increase bone mass.
U.S. Pat. Nos. 5,532,226 and 5,300,687 describe the use of trifluoromethylbenzylphosphonates to increase bone mass.
U.S. Pat. No. 5,885,973 to Papapoulos, et al., discloses a bone mass anabolic composition that includes olpandronate, which is a bisphosphonate.
WO 97/02827 filed by Gador S. A. and the University of Leiden discloses 1-amino-3-(N,N-dimethylamino)-propyliden-1,1-bisphosphonic acid (referred to sometimes as xe2x80x9cIG9402xe2x80x9d) acid as a carrier for bone active substances or for the preparation of a medicament for the diagnosis, prophylaxis and/or mineral metabolism disorders. WO 97/02827 discloses on page 5 that IG9402, illustrated below, is devoid of any antiresorptive activity, which is used as the basis for the essential feature of that disclosed invention. The application does not provide an explanation for why this specific bisphosphonate does not have antiresorptive activity, which has been considered the classic mechanism of action of bisphosphonates.
Estrogens/Progestogens
Estrogens/progestogens (anti-remodeling and anti-resorptive compounds) as a class have not to date been shown to increase bone mass by more than 10%, but instead have been used to retard the effect of osteoporosis. Estrogens are currently the most effective method of preventing osteoporosis in postmenopausal women.
U.S. Pat. No. 5,183,815 discloses the use of a steroidal hormone covalently linked to a hydroxy alkyl-1,1-bisphosphonate.
U.S. Pat. No. 5,843,934 claims that an estrogen having insubstantial sex-related activity can be administered to a patient to retard the adverse effects of osteoporosis in a male or female. The ""934 patent does not address how to select a compound to increase bone mass, but instead teaches how to retard the effect of bone loss.
WO 98/22113 filed by the University of Florida Research Foundation, Inc. discloses methods to utilize an xcex1 or xcex2-isomer of an estrogen compound to confer cytoprotection on a population of cells associated with an ischemic event.
Phytoestrogen
Little is known about the actions of phytoestrogens on bone (Fitzpatrick, L. A., Mayo Clinic Proceedings, 74:601-607, 1999). Soy protein did not prevent increased bone turnover in cynomolgus monkeys; they actually increased it. However, BMD declined after two years in postmenopausal women taking only calcium but did not change in those receiving ipriflavone. Isoflavone significantly increased spinal BMD in postmenopausal women after 6 months of 40 mg/day of soy protein supplementation (containing 90 mg isoflavones) but not with lower doses (56 mg/day) (Feinkel, E. Lancet, 352:762, 1998).
Parathyroid Hormone (PTH)
Daily injections of parathyroid hormone (PTH), an agent known for its role in calcium homeostasis, increases bone mass in animals and humans, as does the related PTH-related hormone PHTrP, the only other known ligand of the PTH receptor. Whereas increased prevalence of apoptosis of osteoblasts and osteocytes are key pathogenic mechanisms for steroid-induced osteoporosis (Weinstein et al., J Clin Invest, 102:274-282, 1998; Weinstein et al., Bone, 23:S461, 1998; Bellido et al., Bone, 23:S324, 1998), the reverse, i.e., postponement of osteoblast apoptosis, is the principal, if not the sole, mechanism for the anabolic effects of intermittent parathyroid hormone administration on bone (Jilka et al., J. Clin. Invest. 104:439-446 1999). The increased bone mineral density, osteoblast perimeter and bone formation rate that occur with intermittent PTH administration in mice happen without a change in osteoblast production. Instead, the anabolic effect of the drug is due to decreased prevalence of osteoblast apoptosis from 1.7-2.2% to as little as 0.1-0.4%, while the osteocytes in the newly made lamellar cancellous bone are closer together and more numerous than those found in the animals receiving vehicle alone. The closely spaced, more numerous osteocytes are the predictable consequence of protecting osteoblasts from apoptosis. The anti-apoptotic effect of PTH on osteoblasts as well as osteocytes has been confirmed in vitro using primary bone cell cultures and established cell lines.
The use of teriparatide (the 1-34 amino acid fragment of human parathyroid growth hormone) to stimulate bone formation has also been investigated; teriparatide administered as daily injections has been reported to selectively increase the trabecular bone density of the spine in osteoporotic patients.
U.S. Pat. No. 5,510,370 discloses the use of a combination of PTH and raloxifene to increase bone mass. U.S. Pat. No. 4,833,125 discloses the use of PTH in combination with either a hydroxylated vitamin D derivative, or a dietary calcium supplement.
Calcium Preparations
Calcium preparations, while useful as a dietary supplement for persons who are calcium deficient, have not been shown effective to increase bone mass. However, they may reduce the rate of bone loss. U.S. Pat. No. 5,618,549 (a calcium salt) describes the use of calcium.
Fluoride
The most thoroughly studied anabolic agent, sodium fluoride, can increase vertebral bone mass by 10% a year for at least four years but there is controversy about the quality of the bone formed. Sodium fluoride has not been approved as a bone anabolic agent. It has been difficult to establish anti-fracture efficacy because of serious qualitative abnormalities. First, much of the new bone is initially woven rather than lamellar. Second and more important, there is severe impairment of bone mineralization, in spite of sodium fluoride""s effectiveness in increasing bone mass.
U.S. Pat. No. 5,071,655 discloses a composition to increase bone mass that includes a fluoride source and a mitogenic hydantoin.
SERMs
SERMs such as tamoxifen and raloxifene have also been used to treat osteoporosis. A recent study carried out with raloxifene indicated that after three years of treatment, women on raloxifene had 30-50% fewer spinal fractures, and had 2-3% increase in bone density in their hips and spine, but showed no fewer nonspinal fractures, a category that includes hip fractures (Ettinger, B., JAMA, 282:637-645, 1999).
U.S. Pat. No. 4,970,237 discloses the use of clomiphene to increase bone mass in premenopausal women.
Vitamin D Derivatives
There have been conflicting reports about the value of Vitamin D or its derivatives on bone loss and bone anabolism. Some studies on the hormonal metabolite of vitamin D, calcitriol, have reported an increase in spinal bone density, but others have found no effect.
The following patents describe the use of Vitamin D derivatives to treat bone disease: U.S. Pat. Nos. 4,973,584; 5,7507,46; 5,593,833; 5,532,391; 5,414,098; 5,403,831; 5,260,290; 5,104,864; 5,001,118; 4,973,584; 4,619,920; and 4,588,716.
Other Compounds
The following patents disclose the use of other compounds for the treatment of bone disease: U.S. Pat. Nos. 5,753,649 and 5,593,988 (azepine derivative); 5,674,844 (morphogen); 5,663,195 (cyclooxygenase-2 inhibitor); 5,604,259 (ibuprofen or flurbiprofen); 5,354,773 (bafilomycine); 5,208,219 (activin); 5,164,368 (growth hormone releasing factor); and 5,118,667, 4,870,054 and 4,710,382 (administration of a bone growth factor and an inhibitor of bone resorption).
U.S. Pat. No. 5,859,001 discloses the use of non-estrogen compounds having a terminal phenol group in a four-ring cyclopentanophenanthrene compound structure to confer neuroprotection to cells.
U.S. Pat. No. 5,824,672 discloses a method for preserving tissues during transplantation procedures that includes administering an effective dose of a cyclopentanophenanthrene compound having a terminal phenol A ring.
WO 98/31381 filed by the University of Florida Research Foundation, Inc. discloses a method for enhancing the cytoprotective effect of polycyclic phenolic compounds on a population of cells that involves the steps of administering a combination of polycyclic phenolic compounds and anti-oxidants to achieve an enhanced effect. One disclosed combination is glutathione and estrogen.
It is an object of the present invention is to provide methods of screening for compounds to increase bone strength.
It is another object of the present invention to provide compounds and compositions to increase bone strength in patients in need thereof.
The present invention is a method and composition to increase bone strength in a manner that decreases fracture incidence, which may or may not include increasing bone mineral density (xe2x80x9cBMDxe2x80x9d). The invention includes administering an effective amount of a bisphosphonate to a host in need thereof to increase bone strength, which inhibits the apoptosis of osteoblasts and osteocytes, without a significant effect on osteoclasts. In one embodiment, the bisphosphonate is not 1-amino-3-(N,N-dimethylamino)-propyliden-1,1-bisphosphonic acid or its pharmaceutically acceptable salt. An increase in osteoblast life span can lead to an increase in bone mass, i.e., an anabolic effect. Preservation of osteocyte life span can increase bone strength, which may be disproportional to the increase in bone mass.
The invention is based on the fundamental discovery that selected bisphosphonates increase bone strength by inhibiting osteocyte and osteoblast apoptosis without substantially affecting osteoclast activity, and thus resorption. In one embodiment, a lack of significant resorption is defined as minimal effect on bone resorption, for example, an effect of decreasing bone resorption by less than 10%, preferably less than 5%, and more preferably less than 2% versus an appropriate control. Lack of significant resorption can be assessed in vitro using the fetal murine long bone assay, or in vivo in ovariectemized mice or postmenopausal women using BMD or biochemical resorption markers (Brown et al., J. Bone Miner. Res. 1998, 13:253-258; van Beek E et al., J. Bone Miner. Res. 1996, 11:1492-1497). In one embodiment, the compound is one other than than 1-amino-3-N,N-dimethylamino)propyliden-1,1-bisphosphonic acid (IG9402), or its pharmaceutically acceptable salt.
Decreased osteoclast activity is associated with decreased remodeling (resorption), which may decrease the quality of bone over time. It was previously thought that the decreased activity of osteoclasts was essential for the activity of bisphosphonates, based on an antiresorption mechanism. This led to the conclusion that bisphosphonates acted as antiresorptives, with possible side effect of decreased bone quality over time. WO 97/02827 disclosed for the first time a bisphosphonate (IG9402) which acts as an anabolic agent without antiresorptive properties. WO 97/02827 did not describe how IG9402 acts as a treatment for osteoporosis without affecting resorption, and therefore, one could not effectively select or design new compounds with these desired properties. This invention presents the fundamental discovery that one essential action of bisphosphonates is the inhibition of apoptosis of osteoblasts and osteocytes, and that the antiresoptive activity (i.e., osteoclast activity) can be decoupled from the apoptosis of osteoblasts and osteocytes. Given this information, one of ordinary skill can select or design a new bisphosphonate which provides these properties as a superior agent to increase bone strength, simply by screening candidate bisphosphonates using identified bone cell assays as described herein or other known methods.
It has also been discovered that the rapid (i.e., within five minutes) activation of ERKs (extracellular signal regulated kinases), which is involved in the inhibition of apoptosis of osteoblasts and osteocytes, can be decoupled from the effect of selected bisphosphonates on osteoclasts and remodeling. Therefore, in another embodiment, a method is presented for treating a host in need of increasing bone strength, that includes selecting or designing a bisphosphonate which causes a rapid activation of ERK in osteoblasts or osteocytes without a significant effect on osteoclasts.
In yet another embodiment of the present invention, there is provided a method of screening for a compound that increases bone strength, that includes the steps of:
1. a) contacting osteocytes with a test compound; b) comparing the number of apoptotic osteocytes treated with the test compound with the number of apoptotic osteocytes not treated with the test compound; and c) determining the effect of the test compound on apoptosis of osteocytes;
2. contacting osteoblasts with the test compound; b) comparing the number of apoptotic osteoblasts treated with the test compound with the number of apoptotic osteoblasts not treated with the test compound; and c) determining the effect of the test compound on apoptosis of osteoblasts; and also
3. contacting osteoclasts with the test compound; b) comparing the number of apoptotic osteoclasts treated with the test compound with the number of apoptotic osteoclasts not treated with the test compound; and c) determining the effect of the test compound on apoptosis of osteoclasts.
In one embodiment, a bisphosphonate which falls within the scope of the present invention is one that causes a significant antiapoptotic effect on osteoblasts and osteocytes (for example, at an in vivo dosage of at least 0.1 ng/kg body weight) or in vitro (at least 10% more apoptotic cells than untreated cells, and preferably, 20, 50 or 75% more apoptotic cells than untreated cells), yet does not have a significant effect on osteoclasts (i.e., less than 10% increase in apoptotic osteoclastic cells, and preferably, less than 5 or 2% apoptotic cells). In another embodment, the test compound induces the phosphorylation of extracellular signal regulated kinase (ERK) (for example, when administered in vivo at a dosage of at least 0.1 ng/kg body weight) or in vitro in osteoblastic or osteocytic cells.
In another embodiment of the present invention, there is provided a method of screening for a compound that prevents glucocorticoid-induced apoptosis of osteocytes without significant bone resorption, comprising the steps of: a) treating osteocytes with a test compound, thereby producing treated osteocytes; b) contacting the treated osteocytes with a glucocorticoid; c) comparing the number of osteocytes undergoing apoptosis in the glucocorticoid-treated osteocytes pretreated with the test compound with the number of osteocytes undergoing apoptosis in the glucocorticoid-treated osteocytes not pretreated with the test compound; and d) determining the effect of the test compound on bone resorption. A lower number of osteocytes undergoing apoptosis in the glucocorticoid-treated osteocytes pretreated with the test compound than the number of osteocytes undergoing apoptosis in the glucocorticoid-treated osteocytes not pretreated with the test compound indicates a test compound that prevents glucocorticoid-induced apoptosis of osteocytes. A compound should be selected that does not have a signficant effect. on bone resorption. Lack of significant resorption can be assessed in vitro using the fetal murine long bone assay, or in vivo in ovariectomized mice or postmenopausal women using BMD or biochemical resorption markers (Brown et al., J. Bone Miner. Res. 1998, 13:253-258; van Beek E et al., J. Bone Miner. Res. 1996, 11:1492-1497).
Evidence is presented herein that bisphosphonates and calcitonin act directly on osteocytes to inhibit apoptosis induced by glucocorticoids or other pro-apoptotic signals. The effect of these agents can be reproduced in osteoblastic cells as well. Significantly, the anti-apoptotic effect of bisphosphonates occurs with doses 100-1000 times lower than those at which these agents inhibit osteoclast resorption. This phenomenon is also demonstrated for bisphosphonates that do not have antiresorptive properties (e.g., IG-9402).
In yet another embodiment of the present invention, there is provided a method of decreasing bone fragility in an individual in need of such treatment, comprising the step of: a) administering to said individual an effective amount of a pharmaceutical composition comprising an amino-bisphosphonate, or salts or hydrates thereof, wherein said administration reduces the number of osteocytes and osteoblasts undergoing apoptosis without significant bone resorption, thereby decreasing bone fragility in said individual.
The present invention indicates that at least part of the anti-fracture efficacy of the bisphosphonate agents for the treatment of osteoporosis is due to the prevention of osteocyte apoptosis. Hence, the present invention discloses in vitro assays of osteocyte and osteoblast apoptosis using osteocytic cell lines that can be used for screening bisphosphonate or calcitonin compounds analogs or other novel agents with anti-fracture properties, thus allowing for the discovery of useful new drugs. The anti-apoptotic efficacy of promising compounds can be subsequently verified in whole animals and human biopsies with techniques which have already been developed for this purpose and have been shown to perform reliably (Weinstein et al., J. Clin. Invest., 102:274-282, 1998).
In the present invention, certain bisphosphonates are used, which, by means of changes in their chemical structure, have reduced anti-osteoclast action. The present invention describes bisphosphonate compound that are capable of reducing the process of osteocytic apoptosis at doses which fail to affect the osteoclast, apparently by a different mechanism of action from which some of the known bisphosphonates act, such that an xe2x80x9costeocyte-selectivexe2x80x9d method of treatment is disclosed herein.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.